Administration of an antagonist of a5ß1 for anti-angiogenesis and cancer treatment

ABSTRACT

The teachings provided herein generally relate to a combination therapy and are directed to pharmaceutical compositions and methods for administering a combination of an a5β1 antagonist with an a2β1 antagonist to a subject. The methods are for use in inhibiting, preventing, or reversing angiogenesis, as well as in treating cancer. In some embodiments, the compositions and methods include a combined administration of VLO4 and VP12 (ECL12).

BACKGROUND

1. Field of the Invention

The teachings provided herein relate to pharmaceutical compositionscomprising an a5β1 antagonist for use in inhibiting angiogenesis andtreating cancer, which can also be used in combination with an a2β1antagonist and in a pharmaceutically acceptable carrier.

2. Description of Related Art

Solid tumor growth is generally considered to be angiogenesis-dependent,such that the control of neovascularization in cancerous tissue is oneof the goals of cancer research. As such, various potential angiogenesisinhibitors have been investigated in the treatment of solid tumors andmetastasis using anti-angiogenic therapy. Unfortunately, however, aparticularly effective method of using an agent or combination of agentsremain to be discovered that, at least, (i) inhibits or preventsangiogenesis; (ii) treats solid tumors to contain and/or reduce tumorsize; and (iii) inhibits or prevents the tumor invasion that leads tometastasis within a subject.

The art is still in need of improved angiogenesis inhibitors, as wellcancer therapies that include such inhibitors. Angiogenesis is a highlyregulated event that involves complex, dynamic interactions betweenmicrovascular endothelial cells and extracellular matrix (ECM) proteins.Control of angiogenesis can be used in a variety of treatments,including cancer therapy. Alteration of ECM composition and architectureis a hallmark of wound clot and tumor stroma. The role of ECM inregulation of angiogenesis associated with wound healing and tumorgrowth still remain generally undefined in the art. During angiogenesis,however, endothelial cell responses to growth factors are modulated bythe compositional and mechanical properties of a surroundingthree-dimensional (3D) extracellular matrix (ECM) that is dominated byeither cross-linked fibrin or type I collagen.

Likewise, a novel method to control tumor invasion to treat a metastaticdisease, for example, would be seen as a significant contribution to theart by one of skill. Over 60% of breast cancer patients have metastaticdisease at diagnosis. The most common cause of death in breast cancerpatients is due to the metastatic spread of the cancer cells from theprimary tumor site to remote sites and growth of the breast cancer cellsat the distant location. Metastasis is a complex process includingseveral mechanisms: (1) migration of the tumor cells through theextracellular matrix surrounding the tumor; (2) invasion of tumor cellsinto angiogenic blood vessels growing into the tumor; (3) adhesion ofthe metastatic cell at a distant site where the microenvironment isreceptive to tumor growth; and (4) newly attached cells must proliferateand induce angiogenesis at the metastatic site. As such, a combinationof select inhibitors could possibly limit this process.

Accordingly, and for at least the above reasons, one of skill willappreciate a method of inhibiting, preventing, or even reversing,angiogenesis. Moreover, one of skill will appreciate a composition andmethod of treatment that can not only inhibit angiogenesis, but that canalso disrupt the physical and mechanical architecture within whichangiogenesis takes place. Such a composition and method may be able to,at least, (i) inhibit or prevent angiogenesis; (ii) treat solid tumorsto contain and/or reduce tumor size; and (iii) inhibit or prevent thetumor invasion that leads to metastasis within a subject.

SUMMARY

The teachings provided herein generally relate to pharmaceuticalcompositions and methods comprising an a5β1 antagonist for use ininhibiting, preventing, or reversing angiogenesis and treating cancerwhen used in combination with an a2β1 antagonist and in apharmaceutically acceptable carrier. In some embodiments, the teachingsare directed to a pharmaceutical formulation comprising an a5β1antagonist, an a2β1 antagonist, and a pharmaceutically acceptablecarrier. The pharmaceutical formulation can, for example; comprise VLO4and VP12 (ECL12). In some embodiments, the teachings are directed to anarticle of manufacture comprising an a5β1 antagonist, an a2β1antagonist, and instructions for administering an effective amount ofthe a5β1 antagonist and an effective amount of the a2β1 antagonist to asubject.

In some embodiments, the methods are directed to inhibiting angiogenesisin a subject, comprising administering an effective amount of an a5β1antagonist in combination with an effective amount of an a2β1 antagonistto the subject. The methods can comprise, for example, administering andeffective amount of VLO4 and VP12 (ECL12) to the subject. In someembodiments, the method further inhibits tumor invasion. And, in someembodiments, the method can inhibit the growth of solid tumors.

In some embodiments, the methods taught herein can further include theadministration of an effective amount of an antiproliferative. And, insome embodiments, the methods can include the administration of aneffective amount of radiation therapy.

In some embodiments, the methods can be directed to inhibitingangiogenesis, inhibiting tumor invasion, inhibiting the growth of solidtumors, or a combination thereof, in a subject. In these embodiments, asdescribed above, the methods can further comprise the administration ofan effective amount of an antiproliferative, an effective amount ofradiation therapy, surgical therapy, or a combination thereof.

VLO4 can be effective when administered alone, or in combination withVP12 (ECL12). The teachings include, for example, a method of reversingangiogenesis in a subject, comprising administering an effective amountof an VLO4 to the subject. In some embodiments the methods includeadministering VLO4 and VP12 (ECL12) to the subject. In some embodiments,the method further inhibits tumor invasion, inhibits angiogenesis,and/or inhibits the growth of solid tumors. Moreover, in someembodiments, the methods can further comprise the administration of aneffective amount of an antiproliferative and/or radiation therapy. Assuch, in some embodiments, the methods further comprise theadministration of an effective amount of chemotherapy.

One of skill reading the teachings that follow will appreciate that theconcepts can extend into additional embodiments that go well-beyond aliteral reading of the claims, the inventions recited by the claims, andthe terms recited in the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrate a study of human microvascular endothelial cellangiogenesis, according to some embodiments.

FIGS. 2A-2F show formation of angiogenic sprouts and capillary tube-likestructures by HDMEC in fibrin stimulated by VEGF (100 ng/ml), accordingto some embodiments.

FIGS. 3A-3D show the presence of multiple cells in capillary tube-likestructure by nuclei staining and presence of lumen demonstrated byconfocal microscopic analysis, according to some embodiments.

FIG. 4 graphically depicts the effect of GPenGRGDSPCA peptides [RGD(VN)]on sprout angiogenesis of HDMEC in fibrin, according to someembodiments.

FIG. 5 graphically depicts the effect of VLO4 disintegrin on sproutangiogenesis of HDMEC in fibrin, according to some embodiments.

FIGS. 6A-6D show micrographs of the inhibitory effect of VLO4disintergrin on sprout angiogenesis of HDMEC in fibrin, according tosome embodiments.

FIGS. 7A and 7B show micrographs of how VLO4 disintergrin disrupts newlyformed angiogenic sprouts HDMEC in fibrin, according to someembodiments.

FIGS. 8A-8D are micrographs showing how VLO4 disintegrin (blockingintegrin alpha5 beta1) and VP12 disintegrin (ECL12, blocking integrinalpha2 beta 1) synergistically inhibited angiogenic sprouts of HDMEC infibrin, according to some embodiments.

FIGS. 9A-9D are micrographs showing that VLO4 disintergrin (blockingintergrin alpha5 beta1) and integrin alpha2 beta1 blocking antibodysynergistically inhibited angiogenic sprouts of HDMEC in fibrin,according to some embodiments.

FIGS. 10A-10D are micrographs showing that Echistatin (blockingintergrin alpha v beta3) and VP12 disintegrin (ECL12, blocking integrinalpha 2 beta 1) synergistically inhibited angiogenic sprouts of HDMEC infibrin, according to some embodiments.

FIG. 11 graphically depicts how VLO4 (3 μg/ml) significantly inhibitsHT1080 tumor cell invasion from contracted collagen into fibrin 3Dmatrices, according to some embodiments.

FIG. 12 graphically depicts how VP12 (3 μg/ml, EC12 blocking integrinalpha2 beta1), VLO4 (3 μg/ml, blocking integrin alpha5 beta1) andEchistatin (3 μg/ml) significantly inhibit HT1080 tumor cell invasionfrom contracted collagen into fibrin 3D matrices, according to someembodiments.

FIG. 13 graphically depicts how VLO4 significantly inhibited H1650(Human metastatic lung cancer cell line) and A549 (human alveolaradenocarcinoma cell line) tumor cell proliferation, according to someembodiments.

DETAILED DESCRIPTION

The teachings provided herein generally relate to pharmaceuticalcompositions and methods comprising an a5β1 antagonist for use ininhibiting, preventing, or reversing angiogenesis and treating cancerwhen used in combination with an a2β1 antagonist and in apharmaceutically acceptable carrier.

In some embodiments, the teachings are directed to a pharmaceuticalformulation comprising an a5β1 antagonist, an a2β1 antagonist, and apharmaceutically acceptable carrier. In some embodiments, the teachingsare directed to an article of manufacture comprising an a5β1 antagonist,an a2β1 antagonist, and instructions for administering an effectiveamount of the a5β1 antagonist and an effective amount of the a2β1antagonist to a subject. In some embodiments, the terms “composition”and “formulation” can be interchangeable.

In some embodiments, the teachings are directed methods of inhibitingangiogenesis in a subject, comprising administering an effective amountof an a5β1 antagonist in combination with an effective amount of an a2β1antagonist to the subject. In some embodiments, the method furtherinhibits tumor invasion. And, in some embodiments, the method caninhibit the growth of solid tumors.

The a5β1 antagonist can include, for example, any chemical moiety thatfunctions to block a5β1. Likewise, the a2β1 antagonist can include, forexample, any chemical moiety that functions to block a2β1. Suchantagonists can include small molecules, such as small moleculepharmaceuticals, or large molecules, such as peptides, oligopeptides,polypeptides, proteins, nucleic acids, oligonucleotides, andpolynucleotides, for example. In some embodiments, the peptide caninclude an RGD-recognition motif. In some embodiments, an antagonist caninclude an antibody such as, for example, a polyclonal antibody or amonoclonal antibody and, in some embodiments, the antibody can behumanized or fully human. The antibody may already be known to bind toan a5β1 antagonist, an a2β1 antagonist, or a combination thereof; or,the antibody can be designed specifically to bind to an a5β1 antagonist,an a2β1 antagonist, or a combination thereof. One of skill will how toselect and/or design an antibody of interest for use with the methodsprovided herein and can appreciate, for example, that there are knownmethods of producing a desired antibody. As such, any inhibitor, orligand, that binds to, and down-regulates, the activity of angiogenesisand/or collagen matrix formation can be used in some embodiments. Suchinhibitors can include, but are not limited to, disintegrins, RGDpeptides, blocking monoclonal antibodies, chemical inhibitors, antisensemRNA, and the like, or any combination thereof.

In some embodiments, the antagonists can include one or more of thedisintegrins that bind to an a5β1 antagonist, an a2β1 antagonist, or acombination thereof. Examples of disintegrins can include disintegrinsobtained from snake venom extracts. Such integrins can include, forexample, RGD and non-RGD, such as KGD, MLD, VGD, and MVD disintegrins,referring to an active peptide sequence which can be, for example, inthe “inhibitory loop” of the sequence.

For example, VLO4 can be effective when administered alone, or incombination with VP12 (ECL12). The teachings include, for example, amethod of reversing angiogenesis in a subject, comprising administeringan effective amount of an VLO4 to the subject. In some embodiments themethods include administering VLO4 and VP12 (ECL12) to the subject. Insome embodiments, the method further inhibits tumor invasion, inhibitsangiogenesis, and/or inhibits the growth of solid tumors. Moreover, insome embodiments, the methods can further comprise the administration ofan effective amount of an antiproliferative and/or radiation therapy. Assuch, in some embodiments, the methods further comprise theadministration of an effective amount of chemotherapy.

The disintegrins from snake venom can comprise (i) a first group ofsingle chain sequence compounds having about 49-51 residues and fourdisulphide bonds; (ii) a second group of single chain sequence compoundshaving about 70 residues and six disulphide bonds; (iii) a third groupof single chain sequence compounds having about 84 residues cross-linkedby seven disulphide bonds; (iv) a fourth group of single chain sequencecompounds having about 100 residues having 16 Cys residues involved informing eight disulphide bonds; and (v) a fifth group of dimericcompounds having homodimers or heterodimers. The dimeric disintegrinscan contain, for example, about 67 residues in each subunit, with tencysteine residues involved in forming four intrachain disulphide bondsand two interchain cysteine linkages.

In some embodiments, the disintegrins can include echistatin, VLO4, VP12(ECL12), or a combination thereof. In some embodiments the disintegrinscan comprise (i) echistatin, eristocophin, eristostatin, andocellatusin; (ii) trigramin, kistrin, flavoridin, albolabrin, andbarbourin; (iii) bitistatin and salmosin 3; (iv) PIII; and (v)contortrostatin, EC3, bilitoxin, and EMF-10; and a combination thereof.And, in some embodiments, the disintegrins can include, for example,EO4, EO5, EMS11, VLO4, VLO5, VB7, VA6, or a combination thereof. U.S.application Ser. No. 12/821,873 is hereby incorporated herein byreference in it's entirety.

In some embodiments, the methods can comprise, for example,administering and effective amount of echistatin and VP12 (ECL12) to thesubject. In some embodiments, contortrostatin may be used in place of,or in addition to, VP12 (ECL12). In some embodiments, an RGD peptidethat blocks a5β1 integrin activity can be used in combination with amonoclonal antibody that blocks a2β1 integrin activity.

The antagonists include proteins, such as the disintegrins. And, anypolypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% identity to one of the proteins taught herein can be used.The term “identity” can be used to refer to the extent to whichsequences are invariant. The identity can be referenced against anentire protein or a defined fragment of the protein, as well as, perhapscombination of protein fragments in the case of a construct of peptidefragments. Computational approaches to sequence alignment are generaleither global or local alignments. Calculating a global alignment is aform of global optimization that “forces” the alignment to span theentire length of all query sequences. By contrast, local alignments canbe used to identify regions of similarity within long sequences that areoften widely divergent overall. Local alignments can be used, but can bemore difficult to calculate because of the additional challenge ofidentifying the regions of similarity. One of skill will appreciate thata variety of computational algorithms are available, including slow butformally optimizing methods like dynamic programming, and efficient, butnot as thorough heuristic algorithms or probabilistic methods designedfor large-scale database search. In some embodiments, the sequencealignments for identity can be local, global, dynamic, progressive,heuristic or probabilistic. And, in some embodiments, the sequencealignments can even be based around the location of a motif of interest,such as an active region of the protein sought for binding. The motif inthe disintegrins, for example, can include the inhibitory loop region,an RGD region, a comparable non-RGD active region, or a combinationthereof. One of skill can readily compare sequence identity for any of anumber of desired purposes such as, for example, assessing thepossibility of binding to a receptor, functioning as an agonist,antagonist, and the like, given the knowledge of the function of a likeprotein or peptide structure. Examples of programs that can be used todetermine identity or homology include, for example, BLAST and FASTA.

A polypeptide can include a variant or mutant of the protein, a chimericconstruct, a fragment, a construct of at least two linked peptidefragments, variants of the fragments, a dimer, and the like. Any of thepolypeptides taught herein can be produced using recombinant proceduresknown to one of skill, or using synthetic procedures in which the aminoacid sequence can be constructed, for example, using liquid or solidphase synthesis techniques, such as Fmoc or Boc methods. Likewise, anyof the polypeptides taught herein can be isolated and/or purified usingprocedures known to one of skill, such as through the use of affinitytags, and the like. In some embodiments, the polypeptides can includethe inhibitory loop regions, RGD or non-RGD peptide fragments, linkersthat include an amino acid, an amino acid sequence, alkylenes, or acombination thereof.

The term “variant” refers to modifications to a peptide that allows thepeptide to retain its binding properties, and such modificationsinclude, but are not limited to, conservative substitutions in which oneor more amino acids are substituted for other amino acids; deletion oraddition of amino acids that have minimal influence on the bindingproperties or secondary structure; conjugation of a linker;post-translational modifications such as, for example, the addition offunctional groups. Examples of such post-translational modifications caninclude, but are not limited to, the addition of modifying groupsdescribed below through processes such as, for example, glycosylation,acetylation, phosphorylation, modifications with fatty acids, formationof disulfide bonds between peptides, biotinylation, PEGylation, andcombinations thereof.

In many embodiments, the molecular weight of an agent should be at orbelow about 40,000 Daltons to ensure elimination of the agent from asubject. In some embodiments, the molecular weight of the agent rangesfrom about 300 Daltons to about 40,000 Daltons, from about 8,000 Daltonsto about 30,000 Daltons, from about 10,000 Daltons to about 20,000Daltons, or any range therein.

The variants can be merely conservatively modified variants of thepolypeptides containing only conservative substitutions. The term“conservatively modified variant” refers to a conservative amino acidsubstitution, which is an amino acid substituted by an amino acid ofsimilar charge density, hydrophilicity/hydrophobicity, size, and/orconfiguration such as, for example, substituting valine for isoleucine.In comparison, a “non-conservatively modified variant” refers to anon-conservative amino acid substitution, which is an amino acidsubstituted by an amino acid of differing charge density,hydrophilicity/hydrophobicity, size, and/or configuration such as, forexample, substituting valine for phenyalanine.

In some embodiments, the methods taught herein can further include theadministration of an effective amount of an additional bioactive agentor therapeutic treatment, such as the administration of an effectiveamount of an antiproliferative and/or an effective amount of radiationtherapy. In some embodiments, the terms “agent” and “therapy” can beinterchangeable. For example, the administration of radiation can beconsidered the administration of a second agent, in some embodiments.

A bioactive agent can be any moiety capable of contributing to atherapeutic effect, a prophylactic effect, both a therapeutic andprophylactic effect, or other biologically active effect in a subject. Abioactive agent can also have diagnostic properties. The bioactiveagents include, but are not limited to, small molecules, nucleotides,oligonucleotides, polynucleotides, amino acids, oligopeptides,polypeptides, and proteins. Bioactive agents can include, but are notlimited to, antiproliferatives, antineoplastics, antimitotics,anti-inflammatories, antiplatelets, anticoagulants, antifibrins,antithrombins, antibiotics, antiallergics, antioxidants, and anyprodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. It is to be appreciatedthat one skilled in the art should recognize that some of the groups,subgroups, and individual bioactive agents may not be used in someembodiments of the present invention.

Antiproliferatives include, for example, actinomycin D, actinomycin IV,actinomycin I1, actinomycin X1, actinomycin C1, and dactinomycin(Cosmegen®, Merck & Co., Inc.). Antineoplastics or antimitotics include,for example, paclitaxel (TAXOL, Bristol-Myers Squibb Co.), docetaxel(TAXOTERE, Aventis S.A.), methotrexate, azathioprine, vincristine,vinblastine, fluorouracil, doxorubicin hydrochloride (ADRIAMYCIN,Pfizer, Inc.) and mitomycin (MUTAMYCIN, Bristol-Myers Squibb Co.), andany prodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. Antiplatelets,anticoagulants, antifibrin, and antithrombins include, for example,sodium heparin, low molecular weight heparins, heparinoids, hirudin,argatroban, forskolin, vapiprost, prostacyclin and prostacyclinanalogues, dextran, D-phe-pro-arg-chloromethylketone (syntheticantithrombin), dipyridamole, glycoprotein IIb/IIIa platelet membranereceptor antagonist antibody, recombinant hirudin, and thrombininhibitors (ANGIOMAX, Biogen, Inc.), and any prodrugs, codrugs,metabolites, analogs, homologues, congeners, derivatives, salts andcombinations thereof. Cytostatic or antiproliferative agents include,for example, angiopeptin, angiotensin converting enzyme inhibitors suchas captopril (CAPOTEN and CAPOZIDE, Bristol-Myers Squibb Co.),cilazapril or lisinopril (PRINVIL and PRINZIDE, Merck & Co., Inc.);calcium channel blockers such as nifedipine; colchicines; fibroblastgrowth factor (FGF) antagonists, fish oil (omega 3-fatty acid);histamine antagonists; lovastatin (MEVACOR, Merck & Co., Inc.);monoclonal antibodies including, but not limited to, antibodies specificfor Platelet-Derived Growth Factor (PDGF) receptors; nitroprusside;phosphodiesterase inhibitors; prostaglandin inhibitors; suramin;serotonin blockers; steroids; thioprotease inhibitors; PDGF antagonistsincluding, but not limited to, triazolopyrimidine; and nitric oxide, andany prodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. Antiallergic agentsinclude, but are not limited to, pemirolast potassium (ALAMAST, Santen,Inc.), and any prodrugs, codrugs, metabolites, analogs, homologues,congeners, derivatives, salts and combinations thereof.

Antibody therapy provides additional bioactive agents that may be usefulwhen administered in combination with the methods taught herein.AVASTATIN, for example, is a human monoclonal antibody to VEGF, hasprovided beneficial results in colorectal cancer, increasing survivaltime by more than 30% when used in combination with the standard Saltzregime of irinotecan, 5-fluorouracil, and leucovorin. One of skill willappreciate that several monoclonal antibodies would be useful, thefollowing providing further examples:

TABLE mAb name Trade name Cancer treated: rituximab RITUXAN non-Hodgkinlymphoma trastuzumab HERCEPTIN breast cancer gemtuzumab MYLOTARG acutemyelogenous leukemia (AML) ozogamicin* alemtuzumab CAMPATH chroniclymphocytic leukemia (CLL) ibritumomab ZEVALIN non-Hodgkin lymphomatiuxetan* tositumomab* BEXXAR non-Hodgkin lymphoma cetuximab ERBITUXcolorectal cancer; head & neck cancers bevacizumab AVASTIN colorectalcancer; non-small cell lung cancer; breast cancer; glioblastoma; kidneycancer panitumumab VECTIBIX colorectal cancer ofatumumab ARZERRA chroniclymphocytic leukemia (CLL) *refers to a conjugated monoclonal antibody

It should be appreciated that, a bioactive agent can be given alone orin combination with other bioactive agents, with the compositions andmethods taught herein. Chemotherapy drugs, for example, are sometimesmost effective when given in combination, as a combination chemotherapyregime. The rationale for combination chemotherapy is to use drugs thatwork by different mechanisms of action, thereby decreasing thelikelihood that resistant cancer cells will develop. When drugs havingdifferent effects are combined, each drug can be used at its optimaldose, sometimes without, and sometimes reducing, intolerable sideeffects.

For some cancers, the best approach may be a combination of surgery,radiation therapy, and/or chemotherapy. Surgery or radiation therapy,for example, treats cancer that is confined locally, while chemotherapycan be used to also kill the cancer cells that have spread to distantsites. Sometimes radiation therapy or chemotherapy can be given beforesurgery to shrink a tumor, thereby improving the opportunity forcomplete surgical removal, making these types of combination therapies apotentially valuable therapy for use with the teachings provided herein,at least in some embodiments. Radiation therapy and low-dosechemotherapy after surgery, for example, can help destroy remainingcancer cells. One of skill will appreciate that the stage of the cancercan be a considerable factor in determining whether single therapy or acombination is desired. For example, early-stage breast cancer may betreated with surgery alone, or by using surgery combined with radiationtherapy, chemotherapy, or a combination thereof, depending on the sizeof the tumor and the risk of recurrence. Locally advanced breast cancer,for example, can be treated with chemotherapy, radiation therapy, andsurgery, in some embodiments.

Any cancer tissue that relies, at least in part, on blood supply tosurvive, may be treatable in some embodiments. Examples of cancers thatmay be treated using the methods taught herein can include, but are notlimited to, prostate, bladder, lung, breast, osteosarcoma, pancreatic,colon, melanoma, testicular, colorectal, urothelial, renal cell,hepatocellular, leukemia, lymphoma, and ovarian cancer and centralnervous system malignancies. Lung cancer, although often disperse, mayalso be treated in some embodiments. Likewise, even liquid cancers, suchas lymphoma and leukemia, including acute myeloid leukemia, may also betreated in some embodiments using methods that incorporate the teachingsprovided herein.

Sometimes the combinations taught herein may not be directed to a curebut, rather, to reduce symptoms and prolong life. Such combinationtherapies can be useful, for example, for subjects having advancedcancers that are not suitable for radiation therapy or surgicaltreatment, such as those having un-resectable non-small cell lungcancer, esophageal cancer, or bladder cancers, for example.

In some embodiments, the methods can comprise, for example,administering an effective amount of VLO4 and an effective amount ofVP12 (ECL12) to the subject, wherein a cytotoxic agent, such as TAXOL,G-CSF, or a combination thereof, for example, can be administered toprovide a combination therapy. And, in some embodiments, these methodscan be accompanied by radiation therapy, surgical therapy, or acombination thereof.

In some embodiments, the methods can be directed to inhibiting orpreventing angiogenesis, reversing angiogenesis, inhibiting orpreventing tumor invasion, inhibiting or preventing the growth of solidtumors, reducing the size of solid tumors, or a combination thereof, ina subject. In these embodiments, as described above, the methods canfurther comprise the administration of an effective amount of anantiproliferative, an effective amount of radiation therapy, surgicaltherapy, or a combination thereof.

One of skill will appreciate that “tumor invasion” can be defined as thepenetration of tissue barriers by migrating cancerous cells, and tumorinvasion can be integral to metastases. Tumor invasion can include theinvasion of surrounding tissue as the tumor grows, and capillaryendothelial cells invade the tumor and create tumor blood vesselsthrough, i.e., neovascularization of the tumor tissue. The tumor cellscan also intravasate into the blood circulation for metastasis. Tumorcells can then arrest into distant organs, extravasate, and againmigrate into the new site and start the invasive cycle again. As such,the inhibition or prevention of tumor invasion can contain a tumor ortumors and inhibit or prevent metastasis.

Uses and Methods of Administration

The compositions can provide a therapeutic and/or prophylactic effect inthe treatment of a disease, or ameliorization of one or more symptoms ofa disease in a subject. The term “subject” and “patient” are usedinterchangeably and refer to an animal such as a mammal including, butnot limited to, non-primates such as, for example, a cow, pig, horse,cat, dog, rat and mouse; and primates such as, for example, a monkey ora human.

The compositions provided herein can be administered to a subject usingany manner of administration known to one of skill. For example, in someembodiments, a localized administration is used and, in some embodimentsa systemic administration is used. In some embodiments, a combination ofsystem and local administration is used. One of skill will appreciatethat the therapeutic program selected, the agents administered, thecondition of the subject, and the effects desired, can affect theadministration schedule and program used.

One of skill understands that the amount of the agents administered canvary according to factors such as, for example, the type of disease,age, sex, and weight of the subject, as well as the method ofadministration. For example, local and systemic administration can callfor substantially different amounts to be effective. Dosage regimens mayalso be adjusted to optimize a therapeutic response. In someembodiments, a single bolus may be administered; several divided dosesmay be administered over time; the dose may be proportionally reduced orincreased; or, any combination thereof, as indicated by the exigenciesof the therapeutic situation and factors known one of skill in the art.It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. Dosage regimens may be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions, and the dosage ranges set forth herein are exemplary onlyand do not limit the dosage ranges that may be selected by medicalpractitioners.

The terms “administration” or “administering” refer to a method ofincorporating a composition into the cells or tissues of a subject,either in vivo or ex vivo to diagnose, prevent, treat, or ameliorate asymptom of a disease. In one example, a compound can be administered toa subject in vivo parenterally. In another example, a compound can beadministered to a subject by combining the compound with cell tissuefrom the subject ex vivo for purposes that include, but are not limitedto, assays for determining utility and efficacy of a composition. Whenthe compound is incorporated in the subject in combination with one oractive agents, the terms “administration” or “administering” can includesequential or concurrent incorporation of the compound with the otheragents such as, for example, any agent described above. A pharmaceuticalcomposition of the invention is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude, but are not limited to, parenteral such as, for example,intravenous, intradermal, intramuscular, and subcutaneous injection;oral; inhalation; intranasal; transdermal; transmucosal; and rectaladministration.

An “effective amount” of a compound of the invention can be used todescribe a therapeutically effective amount or a prophylacticallyeffective amount. An effective amount can also be an amount thatameliorates the symptoms of a disease. A “therapeutically effectiveamount” refers to an amount that is effective at the dosages and periodsof time necessary to achieve a desired therapeutic result and may alsorefer to an amount of active compound, prodrug or pharmaceutical agentthat elicits any biological or medicinal response in a tissue, system,or subject that is sought by a researcher, veterinarian, medical doctoror other clinician that may be part of a treatment plan leading to adesired effect. In some embodiments, the therapeutically effectiveamount may need to be administered in an amount sufficient to result inamelioration of one or more symptoms of a disorder, prevention of theadvancement of a disorder, or regression of a disorder. In someembodiments, for example, a therapeutically effective amount can referto the amount of an agent that provides a measurable response of atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100% ofa desired action of the composition. The term “treating” refers to theadministering one or more therapeutic or prophylactic agents taughtherein.

A “prophylactically effective amount” refers to an amount that iseffective at the dosages and periods of time necessary to achieve adesired prophylactic result such as, preventing, inhibiting, orreversing angiogenesis, tumor growth, or tumor invasion. Typically, aprophylactic dose is used in a subject prior to the onset of a disease,or at an early stage of the onset of a disease, to prevent or inhibitonset of the disease or symptoms of the disease. A prophylacticallyeffective amount may be less than, greater than, or equal to atherapeutically effective amount.

The administration can be local or systemic. In some embodiments, theadministration can be oral. In other embodiments, the administration canbe subcutaneous injection. In other embodiments, the administration canbe intravenous injection using a sterile isotonic aqueous buffer. Inanother embodiment, the administration can include a solubilizing agentand a local anesthetic such as lignocaine to ease discomfort at the siteof injection. In other embodiments, the administrations may beparenteral to obtain, for example, ease and uniformity ofadministration.

The compounds can be administered in dosage units. The term “dosageunit” refers to discrete, predetermined quantities of a compound thatcan be administered as unitary dosages to a subject. A predeterminedquantity of active compound can be selected to produce a desiredtherapeutic effect and can be administered with a pharmaceuticallyacceptable carrier. The predetermined quantity in each unit dosage candepend on factors that include, but are not limited to, (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcreating and administering such dosage units.

A “pharmaceutically acceptable carrier” is a diluent, adjuvant,excipient, or vehicle with which the composition is administered. Acarrier is pharmaceutically acceptable after approval by a state orfederal regulatory agency or listing in the U.S. PharmacopeialConvention or other generally recognized sources for use in subjects.

The pharmaceutical carriers include any and all physiologicallycompatible solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. Examples of pharmaceutical carriers include, but are not limitedto, sterile liquids, such as water, oils and lipids such as, forexample, phospholipids and glycolipids. These sterile liquids include,but are not limited to, those derived from petroleum, animal, vegetableor synthetic origin such as, for example, peanut oil, soybean oil,mineral oil, sesame oil, and the like. Water can be a preferred carrierfor intravenous administration. Saline solutions, aqueous dextrose andglycerol solutions can also be liquid carriers, particularly forinjectable solutions.

Suitable pharmaceutical excipients include, but are not limited to,starch, sugars, inert polymers, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition canalso contain minor amounts of wetting agents, emulsifying agents, pHbuffering agents, or a combination thereof. The compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationscan include standard carriers such as, for example, pharmaceuticalgrades mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. See Martin, E.W.Remington's Pharmaceutical Sciences. Supplementary active compounds canalso be incorporated into the compositions.

In some embodiments, the carrier is suitable for parenteraladministration. In other embodiments, the carrier can be suitable forintravenous, intraperitoneal, intramuscular, sublingual or oraladministration. In other embodiments, the pharmaceutically acceptablecarrier may comprise pharmaceutically acceptable salts.

Pharmaceutical formulations for parenteral administration may includeliposomes. Liposomes and emulsions are delivery vehicles or carriersthat are especially useful for hydrophobic drugs. Depending onbiological stability of the therapeutic reagent, additional strategiesfor protein stabilization may be employed. Furthermore, one mayadminister the drug in a targeted drug delivery system such as, forexample, in a liposome coated with target-specific antibody. Theliposomes can be designed, for example, to bind to a target protein andbe taken up selectively by the cell expressing the target protein.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable for a high drug concentration. In some embodiments, the carriercan be a solvent or dispersion medium including, but not limited to,water; ethanol; a polyol such as for example, glycerol, propyleneglycol, liquid polyethylene glycol, and the like; and, combinationsthereof. The proper fluidity can be maintained in a variety of ways suchas, for example, using a coating such as lecithin, maintaining arequired particle size in dispersions, and using surfactants.

In some embodiments, isotonic agents can be used such as, for example,sugars; polyalcohols that include, but are not limited to, mannitol,sorbitol, glycerol, and combinations thereof; and sodium chloride.Sustained absorption characteristics can be introduced into thecompositions by including agents that delay absorption such as, forexample, monostearate salts, gelatin, and slow release polymers.Carriers can be used to protect active compounds against rapid release,and such carriers include, but are not limited to, controlled releaseformulations in implants and microencapsulated delivery systems.Biodegradable and biocompatible polymers can be used such as, forexample, ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid, polycaprolactone,polyglycolic copolymer (PLG), and the like. Such formulations cangenerally be prepared using methods known to one of skill in the art.

The compounds may be administered as suspensions such as, for example,oily suspensions for injection. Lipophilic solvents or vehicles include,but are not limited to, fatty oils such as, for example, sesame oil;synthetic fatty acid esters, such as ethyl oleate or triglycerides; andliposomes. Suspensions that can be used for injection may also containsubstances that increase the viscosity of the suspension such as, forexample, sodium carboxymethyl cellulose, sorbitol, or dextran.Optionally, a suspension may contain stabilizers or agents that increasethe solubility of the compounds and allow for preparation of highlyconcentrated solutions.

In one embodiment, a sterile and injectable solution can be prepared byincorporating an effective amount of an active compound in a solventwith any one or any combination of desired additional ingredientsdescribed above, filtering, and then sterilizing the solution. Inanother embodiment, dispersions can be prepared by incorporating anactive compound into a sterile vehicle containing a dispersion mediumand any one or any combination of desired additional ingredientsdescribed above. Sterile powders can be prepared for use in sterile andinjectable solutions by vacuum drying, freeze-drying, or a combinationthereof, to yield a powder that can be comprised of the activeingredient and any desired additional ingredients. Moreover, theadditional ingredients can be from a separately prepared sterile andfiltered solution. In another embodiment, the extract may be prepared incombination with one or more additional compounds that enhance thesolubility of the extract.

In some embodiments, a therapeutically or prophylactically effectiveamount of a composition may range in concentration from about 0.001 nMto about 0.10 M; from about 0.001 nM to about 0.5 M; from about 0.01 nMto about 150 nM; from about 0.01 nM to about 500 μM; from about 0.01 nMto about 1000 nM, 0.001 μM to about 0.10 M; from about 0.001 μM to about0.5 M; from about 0.01 μM to about 150 μM; from about 0.01 μM to about500 μM; from about 0.01 μM to about 1000 nM, or any range therein. Insome embodiments, the compositions may be administered in an amountranging from about 0.001 mg/kg to about 500 mg/kg; from about 0.005mg/kg to about 400 mg/kg; from about 0.01 mg/kg to about 300 mg/kg; fromabout 0.01 mg/kg to about 250 mg/kg; from about 0.1 mg/kg to about 200mg/kg; from about 0.2 mg/kg to about 150 mg/kg; from about 0.4 mg/kg toabout 120 mg/kg; from about 0.15 mg/kg to about 100 mg/kg, from about0.15 mg/kg to about 50 mg/kg, from about 0.5 mg/kg to about 10 mg/kg, orany range therein, wherein a human subject is assumed to average about70 kg.

In some embodiments, the compounds can be administered by inhalationthrough an aerosol spray or a nebulizer that may include a suitablepropellant such as, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or acombination thereof. In one example, a dosage unit for a pressurizedaerosol may be delivered through a metering valve. In anotherembodiment, capsules and cartridges of gelatin, for example, may be usedin an inhaler and can be formulated to contain a powderized mix of thecompound with a suitable powder base such as, for example, starch orlactose.

The teachings herein encompass sustained release formulations for theadministration of one or more agents. In some embodiments, the sustainedrelease formulations can reduce the dosage and/or frequency of theadministrations of such agents to a subject.

The compositions can be administered as a pharmaceutical formulation byinjection. In some embodiments, the formulation can comprise the extractin combination with an aqueous injectable excipient. Examples ofsuitable aqueous injectable excipients are well known to persons ofordinary skill in the art, and they, and the methods of formulating theformulations, may be found in such standard references as Alfonso A R:Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton Pa., 1985. Suitable aqueous injectable excipients include water,aqueous saline solution, aqueous dextrose solution, and the like,optionally containing dissolution enhancers for the acid-modifiedarabinogalactan protein composition, such as solution of mannitol orother sugars, or a solution of glycine or other amino acids.

Typically, a composition taught herein can be administered bysubcutaneously, intramuscularly, intraperitoneally, or intravenously,injecting. A localized administration can, in some embodiments, includedirect injection of an agent into the region of the tissue to be treatedsuch as, for example, a solid tumor. In some embodiments, intravenousadministration is used, and it can be continuous intravenous infusionover a period of a few minutes to an hour or more, such as aroundfifteen minutes. The amount administered may vary widely depending onthe type of formulation, size of a unit dosage, kind of excipients, andother factors well known to those of ordinary skill in the art. Theformulation may comprise, for example, from about 0.0001% to about 10%(w/w), from about 0.01% to about 1%, from about 0.1% to about 0.8%, orany range therein, with the remainder comprising the excipient orexcipients.

In some embodiments, the composition can be administered in conjunctionwith at least one other therapeutic agent for the disease state beingtreated, especially another agent capable of treating cancer such as,for example, a chemotherapeutic agent. The amounts of the agents neededcan be reduced, even substantially, such that the amount of the agent oragents required is reduced to the extent that a significant response isobserved from the subject. A significant response can include, but isnot limited to, a reduction or elimination of nausea, a visible increasein tolerance, a faster response to the treatment, a more selectiveresponse to the treatment, or a combination thereof.

The methods can further comprise the administration of an effectiveamount of an antiproliferative, an effective amount of radiationtherapy, surgical therapy, or a combination thereof. The teachings arealso directed to a method of treating a cancer. In some embodiments, themethod comprises administering an agent to a subject in need of a cancertreatment, wherein the dose of the agent is selected to reduce oreliminate an immunosuppression that would otherwise occur whenadministering a substantially higher dose of the agent in the subject;and administering radiation therapy in combination with the agent,wherein the reduction or elimination of the immunosuppression enhancesthe efficacy of the radiation therapy when compared to the efficacy ofthe radiation therapy otherwise observed when administered incombination with the substantially higher dose of the agent in thesubject. In some embodiments, the agent comprises one or morechemotherapeutic agents in combination with the agents provided herein.In these embodiments, the agent can be selected from the groupconsisting of dacarbazine, paclitaxel, doxorubicin, or a combinationthereof.

In some embodiments, an effective amount can range, for example, fromabout 1 mg/day to about 1000 mg/day, from about 10 mg/day to about 500mg/day, from about 50 mg/day to about 250 mg/day, or any range therein,for a human of average body mass. For treating a solid tumor, a similaramount will be therapeutically effective. A person of ordinary skill inthe art will be able without undue experimentation, having regard tothat skill and this disclosure, to determine a therapeutically effectiveamount of the compositions of this invention for a given disease.

In some embodiments, G-CSF is administered in combination with acomposition taught herein using any amount, time, and method ofadministration known to be effective by one of skill. The G-CSF can beNEUPOGEN, for example, administered in an amount ranging from about 0.1μg/kg to about 1 mg/kg, from about 0.5 μg/kg to about 500 μg/kg, fromabout 1 μg/kg to about 250 μg/kg, from about 1 μg/kg to about 100 μg/kgfrom about 1 μg/kg to about 50 μg/kg, or any range therein.

In some embodiments, the radiation therapy can be administered in asingle, localized high-dose ranging, for example, from about 20 Gy toabout 100 Gy. In some embodiments, the radiation therapy can beadministered in a total dose ranging from about 20 Gy to about 100 Gyusing a modified hypofractionation regime of dosing comprising fromabout 2 doses to about 5 doses during a time frame of one week. In someembodiments, the radiation therapy can be administered in a total doseranging from about 20 Gy to about 100 Gy using a modifiedhypofractionation regime of dosing comprising from 2 doses to 3 dosesduring a time frame ranging from about 2 days to about 3 days. Theradiation therapy can also be administered in a total dose ranging fromabout 45 Gy to about 60 Gy using a modified hypofractionation regime ofdosing comprising administering a single dose ranging from about 15 Gyto about 20 Gy for each day during a 3-day time frame.

The compositions and therapies taught herein can be administered incombination. For example, the combinations can be administered, forexample, for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 3 months, 6 months 1year, any combination thereof, or any amount of time considerednecessary by one of skill. The agents can be administered concomitantly,sequentially, or cyclically to a subject. Cycling therapy involves theadministering a first agent for a predetermined period of time,administering a second agent or therapy for a second predeterminedperiod of time, and repeating this cycling for any desired purpose suchas, for example, to enhance the efficacy of the treatment. The agentscan also be administered concurrently. The term “concurrently” is notlimited to the administration of agents at exactly the same time, butrather means that the agents can be administered in a sequence and timeinterval such that the agents can work together to provide additionalbenefit. Each agent can be administered separately or together in anyappropriate form using any appropriate means of administering the agentor agents.

Articles of Manufacture

The present invention provides for articles of manufacture thatencompass finished, packaged and labelled pharmaceutical products. Thearticles of manufacture include the appropriate unit dosage form in anappropriate vessel or container such as, for example, a glass vial orother container that is hermetically sealed. In the case of dosage formssuitable for parenteral administration, the active ingredient, e.g. oneor more agents including an extract taught herein, is sterile andsuitable for administration as a particulate-free solution. In otherwords, the invention encompasses both parenteral solutions andlyophilized powders, each being sterile, and the latter being suitablefor reconstitution prior to injection. Alternatively, the unit dosageform may be a solid suitable for oral, transdermal, topical or mucosaldelivery.

In some embodiments, the unit dosage form is suitable for intravenous,intramuscular, topical or subcutaneous delivery. Thus, the inventionencompasses solutions, which are preferably sterile and suitable foreach route of delivery. The concentration of agents and amountsdelivered are included as described herein.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. In addition, the articles of manufacture can includeinstructions for use or other information material that can advise theuser such as, for example, a physician, technician or patient, regardinghow to properly administer the composition as a prophylactic,therapeutic, or ameliorative treatment of the disease of concern. Insome embodiments, instructions can indicate or suggest a dosing regimenthat includes, but is not limited to, actual doses and monitoringprocedures.

In other embodiments, the instructions can include informationalmaterial indicating that the administering of the compositions canresult in adverse reactions including but not limited to allergicreactions such as, for example, anaphylaxis. The informational materialcan indicate that allergic reactions may exhibit only as mild pruriticrashes or may be severe and include erythroderma, vasculitis,anaphylaxis, Steven-Johnson syndrome, and the like. The informationalmaterial should indicate that anaphylaxis can be fatal and may occurwhen any foreign protein is introduced into the body. The informationalmaterial should indicate that these allergic reactions can manifestthemselves as urticaria or a rash and develop into lethal systemicreactions and can occur soon after exposure such as, for example, within10 minutes. The informational material can further indicate that anallergic reaction may cause a subject to experience paresthesia,hypotension, laryngeal edema, mental status changes, facial orpharyngeal angioedema, airway obstruction, bronchospasm, urticaria andpruritus, serum sickness, arthritis, allergic nephritis,glomerulonephritis, temporal arthritis, eosinophilia, or a combinationthereof.

In some embodiments, the articles of manufacture can comprise one ormore packaging materials such as, for example, a box, bottle, tube,vial, container, sprayer, insufflator, intravenous (I.V.) bag, envelope,and the like; and at least one unit dosage form of an agent comprisingan extract taught herein within the packaging material. In otherembodiments, the articles of manufacture may also include instructionsfor using the composition as a prophylactic, therapeutic, orameliorative treatment for the disease of concern.

In other embodiments, the articles of manufacture can comprise one ormore packaging materials such as, for example, a box, bottle, tube,vial, container, sprayer, insufflator, intravenous (I.V.) bag, envelope,and the like; and a first composition comprising at least one unitdosage form of an agent comprising an extract as taught herein withinthe packaging material, along with a second composition comprising asecond agent such as, for example, a glycosaminoglycan, phospholipid,poly(alkylene glycol), any other bioactive agent taught herein, or anyprodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. In other embodiments, thearticles of manufacture may also include instructions for using thecomposition as a diagnostic, prophylactic, therapeutic, or ameliorativetreatment for the disease of concern.

Without intending to be limited to any theory or mechanism of action,the following examples are provided to further illustrate the teachingspresented herein. It should be appreciated that there are severalvariations contemplated within the skill in the art, and that theexamples are not intended to be construed as providing limitations tothe claims.

Example 1 A Human Model was Prepared to Correlate Angiogenesis with theExtracellular Matrix

In this study, we investigated the correlation between sproutangiogenesis and the integrity of an extracellular matrix (ECM)environment using in vivo and in vitro angiogenesis models. We used ana5β1 antagonist and an a2β1 antagonist in the models, where the a5β1antagonist was the disintegrin VLO4, and the a2β1 antagonist was thedisintegrin VP12 (ECL12).

Materials

A 3-D ECM model was prepared. Gelatin-coated, microcarrier beads(Cytodex-3) were purchased from Pharmacia (Uppsala, Sweden). Sterile,native bovine dermal collagen containing 95% type I collagen and 5% typeIII collagen (Vitrogen) was obtained from Collagen Biomaterials (PaloAlto, Calif.). Dimethyl dichlorosilane, aprotinin, dibutyryl cyclic AMP,hydrocortisone, trypsin, soybean trypsin inhibitor, and EDTA wereobtained from Sigma Chemical Co. (St. Louis, Mo.). Endothelial cellbasal medium (EBM), endothelial cell growth medium bulletkit-2 (EGM-2BULLETKIT), bovine brain extract, and epidermal growth factor wereobtained from Clonetics Corp. (San Diego, Calif.). Normal human serumwas obtained from BioWhittaker, Inc. (Walkersville, Md.). Plateletderived growth factor-BB (PDGF-BB), vascular endothelial cell growthfactor (VEGF) and Basic fibroblast growth factor (bFGF) were purchasedfrom Chemical Co. (St. Louis, Mo.). VEGF-C was kindly provided by KariAlitalo. Human thrombin was obtained from Calbiochem (San Diego,Calif.). And, propidium iodide (PI) was obtained from Molecular Probes(Eugene, Oreg.).

Cell Culture

Human dermal microvascular endothelial cells (HDMEC) were isolated fromhuman neonatal foreskins. Briefly, after initial harvest from mincedtrypsinized human foreskins, microvascular endothelial cells werefurther purified on a PERCOLL density gradient. HDMEC were cultured oncollagen type 1 coated tissue culture flasks in EGM (endothelial cellgrowth medium) consisting of EBM supplemented with 10 ng/ml epidermalgrowth factor, 0.4% bovine brain extract, 17.5 microg/ml dibutyrylcyclic AMP, and 1 microg/ml hydrocortisone in the presence of 30% normalhuman serum. Endothelial cell cultures were characterized and determinedto be >99% pure on the basis of formation of typical cobblestonemonolayers in culture, positive immunostaining for factor VIII-relatedantigen, and selective uptake of acetylated low density lipoprotein. Allexperiments were done with HDMEC below passage 10. Bovine aorticendothelial cells (BAEC) were obtained from BioWhittaker, Inc.(Walkersville, Md.) and cultured according to manufacturer'sinstruction. BAEC between passage 4 and 8 were used for experiments.

Preparation of Endothelial Cell-Loaded Microcarrier Beads (EC-Beads)

Gelatin-coated CYTODEX-3 microcarrier beads were prepared as describedby the manufacturer. Approximately 80,000 sterile microcarrier beadswere washed, resuspended in EGM, and added to approximately 4.5 millionendothelial cells (HDMEC or BAEC). The beads and cells were mixed bygentle swirling, incubated at 37° C. for 6 hr, and then rotated for24-36 hr on an orbital mixer in a 37° C. oven to generate endothelialcell-loaded microcarrier beads (EC-beads).

Example 2 Cell Migration and Capillary Sprout Formation was Identifiedin Fibrin Gels and Type I Collagen Gels in the Human Model

FIGS. 1A-1C illustrate a study of human microvascular endothelial cellangiogenesis, according to some embodiments. A microcarrier, in vitroangiogenesis assay, previously designed to investigate bovine pulmonaryartery endothelial cell angiogenic behavior in bovine fibrin gels, wasmodified for the study of human microvascular endothelial cellangiogenesis. The HDMEC were isolated from human neonatal foreskins andused, as described above, and images were captured at variousmagnifications, where the effect of angiogenic factors on sproutangiogenesis was quantified visually by counting the number and percentof EC-beads with capillary sprouts.

FIG. 1A shows the process in Step I, where human fibrinogen, isolated aspreviously described, was dissolved in M199 medium at a concentration of1 mg/ml (pH 7.4) and sterilized by filtering through a 0.22 micronfilter. An isotonic 1.5 mg/ml collagen solution was prepared by mixingsterile native bovine type I & III collagen (Vitrogen, CollagenBiomaterials, Palo Alto, Calif.) in 5XM199 medium and distilled water.The pH was adjusted to 7.4 using 1N NaOH.

FIG. 1A also shows the process in Step II, where in certain experiments,growth factors, such as VEGF, VEGF-C, bFGF or PDGF-BB, were added to thefibrinogen and collagen solutions.

For experiments using RGD peptides, EC-beads were firstly incubated withvary concentrations of peptides for one hour and then were added to theECM solutions. About 500 EC-beads were then added to the ECM proteinsolutions, followed by the addition of 0.5 U/ml human thrombin. A 0.3 mlaliquot of each suspension was immediately added to appropriate wells ofa 24-well tissue culture plate. After gelation, 1 ml of fresh assaymedium (EBM supplemented with 20% normal human serum for HDMEC or EBMsupplemented with 10% fetal bovine serum for BAEC) was added to eachwell.

FIGS. 1B and 1C show how the angiogenic response was monitored visuallyand recorded by video image capture. Specifically, capillary sproutformation was observed and recorded with a NIKON Diaphot-TMD invertedmicroscope (Nikon Inc., Melville, N.Y.), equipped with an incubatorhousing with a NIKON NP-2 thermostat and Sheldon #2004 carbon dioxideflow mixer. The microscope was directly interfaced to a video systemconsisting of a Dage-MTI CCD-72S video camera and Sony 12″ PVM-122 videomonitor linked to a Macintosh G3 computer. The images were captured atvarious magnifications using Adobe Photoshop. The effect of angiogenicfactors on sprout angiogenesis was quantified visually by determiningthe number and percent of EC-beads with capillary sprouts. 100-200 beads(five random low power fields) in each of triplicate wells were countedfor each experimental condition. All experiments were repeated at leastthree times. FIG. 1B shows an absence of sprout angiogenesis usinganti-angiogenic factor, and FIG. 1C shows the presence of sproutangiogenesis using angiogenic factor.

FIGS. 2A-2F show formation of angiogenic sprouts and capillary tube-likestructures by HDMEC in fibrin stimulated by VEGF (100 ng/ml), accordingto some embodiments. FIG. 2A illustrates what occurs in control fibringel without addition of angiogenic factors, where no significant sproutformation occurred after 48 hr. FIG. 2B illustrates that, in thepresence of VEGF, HDMEC formed angiogenic sprouts and invaded andmigrated into the fibrin gel within 48 hr. FIG. 2C shows that, by 5days, VEGF-stimulated HDMEC formed branching capillary tube-likestructures in the fibrin gel. FIG. 2D shows formation of local capillaryarcades and networks by HDMEC in fibrin with VEGF for 5 days. FIG. 2Eshows formation of wild capillary networks by HDMEC in fibrin with VEGFfor 5 days. FIG. 2F, a higher magnification of a typical capillarynetwork, illustrates that networks are formed as a result of branchingand fusion of capillary tubes from neighboring beads. a) and b) 100×, c)and d) 400× magnification

FIGS. 3A-3D show the presence of multiple cells in capillary tube-likestructure by nuclei staining and presence of lumen demonstrated byconfocal microscopic analysis, according to some embodiments. FIG. 3Ashows HDMEC formed elongated and branched capillary sprouts in fibringel after 5 days in presence of VEGF at 100 ng/ml. In FIG. 3B, theculture in a) was fixed and stained with Propidium iodide (PI) to revealnuclei. FIG. 3C shows a phase-contrast photomicrograph of typicalcapillary tube-like structure formed by HDMEC in fibrin with 100 ng/mlVEGF. FIG. 3D shows a series of contiguous 1 micron thick tangentialcross sections of the capillary tube-like structure, obtained byreflective confocal microscopy with computerized imaging, initiallyrevealed the top of the tube (0-3 microns) and then the presence of acentral lumen (8-19 microns) between two walls. a) and b) 100×, c) andd) 400× magnification.

FIG. 4 graphically depicts the effect of GPenGRGDSPCA peptides [RGD(VN)]on sprout angiogenesis of HDMEC in fibrin, according to someembodiments. GPenGRGDSPCA (SEQ ID NO:5) is a specificarginine-glycine-aspartic acid (RGD) antagonist HDMEC cultured onmicrocarrier beads were incubated with various doses of RGD[VN] (12.5mM-100 mM) peptides at RT for 1 hr. The EC-beads were added tofibrinogen solution with presence of VEGF (100 ng/ml) and thenpolymerized with thrombin. RGD[VN] significantly inhibited sproutangiogenesis of HDMEC in fibrin compared to GRGESP[RGE] hexapeptide (SEQID NO:6) control peptides.

FIG. 5 graphically depicts the effect of VLO4 disintegrin on sproutangiogenesis of HDMEC in fibrin, according to some embodiments. HDMECcultured on microcarrier beads were incubated with various doses of VLO4(0.3 μg/ml-3 μg/ml) at RT for 30 minutes. The EC-beads were added tofibrinogen solution with presence of VEGF (100 ng/ml) and thenpolymerized with thrombin. VLO4 dose-dependently inhibits sproutangiogenesis of HDMEC induced by VEGF in fibrin.

FIGS. 6A-6D show micrographs illustrating the effect of VLO4 disintegrinon sprout angiogenesis of HDMEC in fibrin, according to someembodiments. HDMEC cultured on microcarrier beads were incubated withvarious doses of VLO4 (0.01 μg/ml, 0.1 μg/ml, 0.3 μg/ml, and 1 μg/ml,FIGS. 6A-6D, respectively) at room temperature for 30 minutes. TheEC-beads were added to fibrinogen solution with presence of VEGF (100ng/ml) and then polymerized with thrombin. VLO4 dose-dependentlyinhibits sprout angiogenesis of HDMEC induced by VEGF in fibrin.

FIGS. 7A and 7B show micrographs of how VLO4 disintergrin disrupts newlyformed angiogenic sprouts HDMEC in fibrin, according to someembodiments. HDMEC cultured on microcarriers beads were mixed with VEGF(30 ng/ml), bFGF (25 ng/ml) and heparin like polysaccharides CH2 (10μg/ml) various doses of VLO4 (0.01 μg/ml-1 μg/ml). The EC-beads wereadded to fibrinogen solution and then polymerized with thrombin. FIG. 7Ashows that HDEMC formed extensive capillary sprouts in fibrin after 5days. FIG. 7B shows that, where VLO4 (3 μg/ml) was added into the systemon day 5, it totally disrupted the newly formed sprout angiogenesiswithin 24 hours.

FIGS. 8A-8D are micrographs showing how VLO4 disintegrin (blockingintegrin alpha5 beta1) and VP12 disintegrin (ECL12, blocking integrinalpha2 beta 1) synergistically inhibited angiogenic sprouts of HDMEC infibrin, according to some embodiments. HDMEC cultured on microcarriersbeads were mixed with VEGF (30 ng/ml), bFGF (25 ng/ml) with or withoutVLO4 (0.01 μg/ml) and/or VP12 (10 μg/ml). The EC-beads were added tofibrinogen solution and then polymerized with thrombin. FIG. 8A showsthat HDEMC formed extensive capillary sprouts of HDMEC induced by VEGFand bFGF in fibrin after 3 days.

FIG. 8B shows that low doses of VLO4 (0.01 μg/ml) didn't significantlyinhibit sprout angiogenesis of HDMEC. FIG. 8C shows that VP12 (10 μg/ml)didn't significantly inhibit sprout angiogenesis. FIG. 8D show that acombination of VLO4 and VP12 synergistically inhibited sproutangiogenesis in fibrin induced by VEGF and bFGF.

FIGS. 9A-9D are micrographs showing that VLO4 disintergrin (blockingintergrin alpha5 beta1) and integrin alpha2 beta1 blocking antibodysynergistically inhibited angiogenic sprouts of HDMEC in fibrin,according to some embodiments. HDMEC cultured on microcarrier beads weremixed with VEGF (30 ng/ml), bFGF (25 ng/ml) with or without VLO4 (0.01μg/ml) and/or integrin alpha2 beta1 blocking antibody (5 μg/ml). TheEC-beads were added to fibrinogen solution and then polymerized withthrombin. FIG. 9A shows that HDEMC formed extensive capillary sprouts ofHDMEC induced by VEGF and bFGF in fibrin after 3 days. FIG. 9B showsthat low doses of VLO4 (0.01 μg/ml) didn't significantly inhibit sproutangiogenesis of HDMEC. FIG. 9C shows that Integrin alpha2 beta1 blockingantibody didn't significantly inhibit sprout angiogenesis. FIG. 9D showsthat a combination of VLO4 and integrin alpha2 beta1 blocking antibodysynergistically inhibited sprout angiogenesis in fibrin induced by VEGFand bFGF.

FIGS. 10A-10D are micrographs showing that Echistatin (blockingintergrin alpha v beta3) and VP12 disintegrin (ECL12, blocking integrinalpha 2 beta 1) synergistically inhibited angiogenic sprouts of HDMEC infibrin, according to some embodiments. HDMEC cultured on microcarriersbeads were mixed with VEGF (30 ng/ml), bFGF (25 ng/ml) with or withoutechistatin (0.1 μg/ml) and/or VP12 (10 μg/ml). The EC-beads were addedto fibrinogen solution and then polymerized with thrombin. FIG. 10Ashows that HDEMC formed extensive capillary sprouts of HDMEC induced byVEGF and bFGF in fibrin after 3 days. FIG. 10B shows that sow doses ofechistatin (0.1 μg/ml) didn't significantly inhibit sprout angiogenesisof HDMEC. FIG. 10C shows that VP12 (10 μg/ml) didn't significantlyinhibit sprout angiogenesis FIG. 10D shows that a combination ofechistatin and VP12 synergistically inhibited sprout angiogenesis infibrin induced by VEGF and bFGF.

Example 3 Analysis of an HT1080 Tumor Model

FIG. 11 graphically depicts how VLO4 (3 μg/ml) significantly inhibitsHT1080 tumor cell invasion from contracted collagen into fibrin 3Dmatrices, according to some embodiments. VlO4 dose dependently inhibitsHT1080 tumor cells invade from contracted 3D collagen matrices into 3Dfibrin matrices.

FIG. 12 graphically depicts how VP12 (3 μg/ml, EC12 blocking integrinalpha2 beta1), VLO4 (3 μg/ml, blocking integrin alpha5 beta1) andEchistatin (3 μg/ml) significantly inhibit HT1080 tumor cell invasionfrom contracted collagen into fibrin 3D matrices, according to someembodiments. It indicates that integrin alpha V beta 3, integrin alpha 2beta 1 and integrin alpha 5 beta 1 all play important role in tumorinvasion.

FIG. 13 graphically depicts how VLO4 significantly inhibited H1650(Human metastatic lung cancer cell line) and A549 (human alveolaradenocarcinoma cell line) tumor cell proliferation, according to someembodiments. It indicates that VLO4 not only inhibits tumor invasion, italso inhibits tumor cell profliferation.

Example 4 Fibrin and Collagen Receptors Synergistically RegulateAngiogenesis

We've shown that integrin β1 expression is highly up-regulated in fibrinrich, but not in collagen rich, matrix environments in vitro and invivo. The 3D HDMEC model described above was used in this experimentthat demonstrates combination therapy using two disintegrins, VLO4 andVP12 (ECL12).

Disintegrins represent a novel family of integrin β1 and β3 inhibitorproteins isolated from viper venoms. They are low molecular-weight,cysteine-rich peptides containing the Arg-Gly-Asp (RGD) sequence. Theyare the most potent known inhibitors of integrin function. Disintegrinsinterfere with cell adhesion to the extracellular matrix, includingadhesion of melanoma cells and fibroblasts to fibronectin, and arepotent inhibitors of platelet aggregation.

VLO4 is derived from the venom of Vipera lebetina obtusa. It is a potentirreversible a5β1 integrin antagonist. The following structure is arepresentation of VLO4, a 65 amino acid structure:

VLO4 can be requested, for example, from Sigma-aldrich. See CAS No.105718. See also, Calvete, J. Biochem. J. 372:725-734 (2003), which ishereby incorporated by reference herein in its entirety.

VP12 (ECL12) (Vipera paleastinae venom or VP12) isolated from Viperapaleastinae venom showed a potent inhibitory activity against collagenreceptors a2β1 integrins. Structurally, VP12 is composed of two subunitsVP12A and VP12B displaying amino acid sequence homology withheterodimeric C-lectin type proteins. Sigma Aldrich offers Viperapaleastinae venom under catalog no. V0628, and the composition used forthis example was graciously received from Dr. Cezary Marcinkiewicz,Biotechnology Center, Temple University College of Science andTechnology, Philadelphia, Pa. 19122. See Cancer Biol Ther. 8(15):1507-16 (2009), which is hereby incorporated by reference herein inits entirety.

(SEQ ID NO: 2)

The VP12 (ECL12) subunits VP12A and VP12B are shown, where the grayareas represent conserved amino acids, gaps (−) were included tomaximize sequence similarities, and the X represents unidentified aminoacids in the VP12A subunit. See also, Staniszewska, I. Cancer Biology &Therapy 8 (15):1507-1516 (2009), which is hereby incorporated byreference herein in its entirety.

FIGS. 7A-7D show synergistic inhibition of sprout angiogenesis whencombining echistatin, an avβ3 antagonist with VP12 (ECL12), an a2β1agonist, according to some embodiments. FIG. 7A shows sproutangiogenesis in a HDMEC culture in VEGF and bFGF. FIG. 7B shows theinhibition of sprout angiogenesis when echistatin is added to theculture of FIG. 7A. FIG. 7C shows the inhibition of sprout angiogenesiswhen VP12 (ECL12) is added to the culture of FIG. 7A. FIG. 7D, however,shows the synergistic inhibition of sprout angiogenesis when bothechistatin and VP12 (ECL12) are added to the culture of FIG. 7A.

Echistatin, a disintegrin specific for avβ3, dose dependently inhibitssprout angiogenesis of HDMEC in fibrin. While VP12 (ECL12), adisintegrin inhibitor to collagen receptor integrin a2β1, has noinhibitory effect on sprout angiogenesis in vitro. Surprisingly,however, this example illustrates how VP12 (ECL12) significantly andsynergistically enhanced the inhibition of sprout angiogenesis byechistatin when administered in combination with echistatin Echistatin(Disintegrin echistatin-alpha or Carinatin) is derived from the venom ofEchis carinatus (Saw-scaled viper). It is a potent irreversible avβ3integrin antagonist that disrupts attachment of osteoclasts to bone andinhibits bone reabsorption, prevents ADP-induced platelet aggregationvia inhibition of glycoprotein IIb/IIIa (GpIIb/IIIa, aIIbβ3) receptors.Echistatin inhibits fibrinogen interaction with platelet receptorsexpressed on the glycoprotein IIb-IIIa complex, acts by binding to theglycoprotein IIb-IIIa receptor on the platelet surface, and inhibitsaggregation induced by ADP, thrombin, platelet-activating factor andcollagen. The following structure is a representation of echastatin, a49 amino acid structure for echistatin:

(SEQ ID NO: 4) 1       10         20         30         40QCESGPCCRN CKFLKEGTIC KRARGDDMDD YCNGKTCDCP        49 RNPHKGPAT

Echistatin is commercially available, for example, from Sigma-aldrich.See CAS Nos. 129038-42-2 and 154303-05-6. See also, Calvete, J. Biochem.J. 372:725-734 (2003), which is hereby incorporated by reference hereinin its entirety.

1-3. (canceled)
 4. A method of inhibiting angiogenesis in a subject,comprising administering an effective amount of an α5β1 antagonist incombination with an effective amount of an α2β1 antagonist to thesubject.
 5. The method of claim 4, comprising administering VLO4 andVP12 (ECL12) to the subject.
 6. The method of claim 4, wherein themethod further inhibits tumor invasion.
 7. The method of claim 4,wherein the method inhibits the growth of solid tumors.
 8. The method ofclaim 4, further comprising the administration of an effective amount ofan antiproliferative.
 9. The method of claim 4, further comprising theadministration of an effective amount of radiation therapy.
 10. Themethod of claim 4, further comprising the administration of an effectiveamount of chemotherapy.
 11. A method of inhibiting angiogenesis in asubject, comprising administering an effective amount of VLO4 incombination with an effective amount of VP12 (ECL12) to the subject. 12.The method of claim 11, wherein the method further inhibits tumorinvasion.
 13. The method of claim 11, wherein the method furtherinhibits the growth of solid tumors.
 14. The method of claim 11, furthercomprising the administration of an effective amount of anantiproliferative.
 15. The method of claim 11, further comprising theadministration of an effective amount of radiation therapy.
 16. Themethod of claim 11, further comprising the administration of aneffective amount of chemotherapy.
 17. A method of inhibiting the growthof a solid tumor in a subject, comprising administering an effectiveamount of VLO4 in combination with an effective amount of VP12 (ECL12)to the subject.
 18. The method of claim 17, wherein the method furtherinhibits tumor invasion.
 19. The method of claim 17, further comprisingthe administration of an effective amount of an antiproliferative. 20.The method of claim 17, further comprising the administration of aneffective amount of radiation therapy.
 21. The method of claim 17,further comprising the administration of an effective amount ofchemotherapy.
 22. A method of inhibiting tumor invasion in a subject,comprising administering an effective amount of VLO4 in combination withan effective amount of VP12 (ECL12) to the subject.
 23. The method ofclaim 22, wherein the method inhibits the growth of solid tumors. 24.The method of claim 22, further comprising the administration of aneffective amount of an antiproliferative.
 25. The method of claim 22,further comprising the administration of an effective amount ofradiation therapy.
 26. The method of claim 22, further comprising theadministration of an effective amount of chemotherapy.
 27. A method ofreversing angiogenesis in a subject, comprising administering aneffective amount of VLO4 to the subject.
 28. The method of claim 27,comprising administering VLO4 and VP12 (ECL12) to the subject.
 29. Themethod of claim 27, wherein the method further inhibits tumor invasion.30. The method of claim 27, wherein the method inhibits the growth ofsolid tumors.
 31. The method of claim 27, further comprising theadministration of an effective amount of an antiproliferative.
 32. Themethod of claim 27, further comprising the administration of aneffective amount of radiation therapy.
 33. The method of claim 27,further comprising the administration of an effective amount ofchemotherapy.
 34. A method of inhibiting angiogenesis in a subject,comprising administering an effective amount of VLO4 to the subject. 35.The method of claim 34, wherein the method further inhibits tumorinvasion.
 36. The method of claim 34, wherein the method furtherinhibits the growth of solid tumors.
 37. The method of claim 34, furthercomprising the administration of an effective amount of anantiproliferative.
 38. The method of claim 34, further comprising theadministration of an effective amount of radiation therapy.
 39. Themethod of claim 34, further comprising the administration of aneffective amount of chemotherapy.
 40. A method of inhibiting the growthof a solid tumor in a subject, comprising administering an effectiveamount of VLO4 to the subject.
 41. The method of claim 40, wherein themethod further inhibits tumor invasion.
 42. The method of claim 40,further comprising the administration of an effective amount of anantiproliferative.
 43. The method of claim 40, further comprising theadministration of an effective amount of radiation therapy.
 44. Themethod of claim 40, further comprising the administration of aneffective amount of chemotherapy.
 45. A method of inhibiting tumorinvasion in a subject, comprising administering an effective amount ofVLO4 to the subject.
 46. The method of claim 45, wherein the methodinhibits the growth of solid tumors.
 47. The method of claim 45, furthercomprising the administration of an effective amount of anantiproliferative.
 48. The method of claim 45, further comprising theadministration of an effective amount of radiation therapy.
 49. Themethod of claim 45, further comprising the administration of aneffective amount of chemotherapy.
 50. A method of inhibitingangiogenesis in a vascular tissue, comprising contacting a vascular celltissue with a composition comprising an α5β1 antagonist and an α2β1antagonist.
 51. The method of claim 50, the composition comprising VLO4and VP12 (ECL12).
 52. A method of inhibiting angiogenesis in a vasculartissue, comprising contacting a vascular cell tissue with a compositioncomprising VLO4.